Anaerobic killing of mucoid Pseudomonas aeruginosa by acidified nitrite derivatives under cystic fibrosis airway conditions
Sang Sun Yoon, … , Richard C. Boucher, Daniel J. Hassett
Sang Sun Yoon, … , Richard C. Boucher, Daniel J. Hassett
Published February 1, 2006
Citation Information: J Clin Invest. 2006;116(2):436-446. https://doi.org/10.1172/JCI24684.
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Research Article Pulmonology

Anaerobic killing of mucoid Pseudomonas aeruginosa by acidified nitrite derivatives under cystic fibrosis airway conditions

Abstract

Mucoid, mucA mutant Pseudomonas aeruginosa cause chronic lung infections in cystic fibrosis (CF) patients and are refractory to phagocytosis and antibiotics. Here we show that mucoid bacteria perish during anaerobic exposure to 15 mM nitrite (NO2–) at pH 6.5, which mimics CF airway mucus. Killing required a pH lower than 7, implicating formation of nitrous acid (HNO2) and NO, that adds NO equivalents to cellular molecules. Eighty-seven percent of CF isolates possessed mucA mutations and were killed by HNO2 (3-log reduction in 4 days). Furthermore, antibiotic-resistant strains determined were also equally sensitive to HNO2. More importantly, HNO2 killed mucoid bacteria (a) in anaerobic biofilms; (b) in vitro in ultrasupernatants of airway secretions derived from explanted CF patient lungs; and (c) in mouse lungs in vivo in a pH-dependent fashion, with no organisms remaining after daily exposure to HNO2 for 16 days. HNO2 at these levels of acidity and NO2– also had no adverse effects on cultured human airway epithelia in vitro. In summary, selective killing by HNO2 may provide novel insights into the important clinical goal of eradicating mucoid P. aeruginosa from the CF airways.

Authors

Sang Sun Yoon, Ray Coakley, Gee W. Lau, Sergei V. Lymar, Benjamin Gaston, Ahmet C. Karabulut, Robert F. Hennigan, Sung-Hei Hwang, Garry Buettner, Michael J. Schurr, Joel E. Mortensen, Jane L. Burns, David Speert, Richard C. Boucher, Daniel J. Hassett

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Figure 2

Mucoid P. aeruginosa FRD1 is selectively killed by NO2– in a pH-dependent manner.

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Mucoid P. aeruginosa FRD1 is selectively killed by NO2– in a pH-dependen...
Anaerobic growth of P. aeruginosa strains at pH 6.5 using 15 mM NO3– or NO2– as a terminal electron acceptor. Aerobic overnight suspensions of PAO1, FRD1, and FRD1/pmucA were diluted 100-fold for the main anaerobic culture with NO3– (A) or NO2– (B). CFUs were enumerated each day and plotted as linear (A) and logarithmic (B) graphs. (C) NO2– sensitivity versus pH. FRD1 or FRD1/pmucA were seeded onto LB agar buffered at the indicated pH value. After placing a filter disk containing 10 μl of 1 M NO2–, the plates were incubated anaerobically for 48 hours and scanned for viewing the zone of killing. To support anaerobic growth, 15 mM NO3– was included in the media. (D) Mucoid strain FRD1 (gray bars) and nonmucoid FRD1/pmucA (black bars) were incubated together at the indicated ratios (FRD1/pmucA:FRD1) for 5 days in the presence of 15 mM NO2–, pH 6.5, after which CFUs were determined. *P < 0.001 versus CFU in the initial inoculum. (E) Dose-response killing of mucoid strain FRD1 by NO2–. Bacteria were suspended in LB (pH 6.5) with various amounts of NO2– for 24 hours under anaerobic conditions. Survival against NO2– is presented as the percentage of CFU relative to that in initial inoculum. (F) Long-term anaerobic exposure of mucoid strain FRD1 to 15 mM NO2– at pH 6.5. All experiments were performed in triplicate and presented as mean ± SEM.